Low molecular weight compounds responsible for savory taste of Indonesian soy sauce

Indonesian soy sauce is made using only soybeans as the nitrogenous source. Moromi obtained from fermentation of yellow soybeans using Aspergillus sojae as the starter was investigated. The fraction with molecular weights of less than 500 Da obtained by stepwise ultrafiltration was then fractionated by several chromatographic procedures, including gel filtration chromatography and RP-HPLC. Several chemical analyses, CE profiles, and taste profiles were performed to obtain the most intense umami fraction. The main components eliciting or enhancing the umami taste present in the fraction were purified and identified by protein sequencing, ESI-MS, and (1)H NMR at 400 MHz. Besides free l-glutamic acid and aspartic acid, free aromatic amino acids such as l-phenylalanine and l-tyrosine may also play an important role in impressing savory or umami taste of Indonesian soy sauce at their subthreshold concentrations and in the presence of salt and free acidic amino acids. This is reported as a new phenomenon of the so-called bitter amino acids.     

(13)C nuclear magnetic resonance spectra of natural undiluted lipids: docosahexaenoic-rich phospholipid and triacylglycerol from fish

In the (13)C NMR spectra of natural, unenriched docosahexaenoic acid-rich sardine oil and cod muscle glycerophosphocholine significant signal intensity differences across the 1D spectrum between undiluted and diluted samples were observed. In undiluted samples (13)C-(13)C 2D nuclear Overhauser enhancement spectroscopy (NOESY) interchain cross-peaks for CH, CH(2), CH(3), and C=C structures were observed. Results indicate that in undiluted natural lipids, NMR signal intensity is influenced by polarization transfer from the extended lipid structure. The NOE enhancement of specific molecular sites especially in unsaturated lipids is evidence that some natural lipids remain oriented relative to each other and in an orderly arrangement at the molecular level long enough for the effect to be detected by the NMR experiment. The presence of polyunsaturated fatty acids in mixtures of natural lipids could stabilize specific local molecular conformations within the remaining less saturated lipids.     

Raman spectral analysis in the C-H stretching region of proteins and amino acids for investigation of hydrophobic interactions.

Raman spectra of amino acids showed complexity in the C-H stretching region (2800-3100 cm(-)(1)) attributed to diversity of CH, CH(2), and CH(3) groups in the side chains, ionization state, and microenvironment. The involvement of specific amino acids in the C-H stretching region of selected proteins, namely, lysozyme, alpha-lactalbumin, beta-lactoglobulin, and their binary mixtures, was investigated by deconvolution using maximum likelihood techniques. The main protein band near 2940 cm(-)(1) was attributed not only to aromatic and aliphatic amino acids but also to many other amino acids. A band near 3065 cm(-)(1) was assigned to aromatic residues, whereas bands near 2880 and 2900 cm(-)(1) corresponded primarily to aliphatic amino acids. Heating at 90 degrees C increased relative intensity near 2940 cm(-)(1) and decreased relative intensity at 2895-2902 cm(-)(1) for lysozyme and its mixtures with alpha-lactalbumin or beta-lactoglobulin. Additional bands at 2812 or 2838 and 3003 cm(-)(1) were observed after heating or in 8 M deuterated urea, reflecting changes upon denaturation.     

Differentiation of extractive and synthetic salicin. The 2H aromatic pattern of natural 2-hydroxybenzyl alcohol

The natural abundance deuterium NMR characterization of four commercially available samples (Kahlbaum, Aldrich, Fluka, and Extrasynthese) of salicin 1 in comparison with two extractive samples from Salix humboldiana and Salix purpurea L. and with a synthetic material, performed on the pentaacetate derivative 2 and on diacetyl salicyl alcohol 4, is reported. Product 2 from S. humboldiana and the sample from salicin Kahlbaum show mean (D/H)aromatic values of 117 and 121 ppm, whereas, for the remaining, values of 146, 154, 153, and 150 ppm are observed, thus suggesting that salicin Kahlbaum is from extractive origin. The (D/H) values at positions 5' and 6' of the sugar moiety suggest a hypothesis on the origin of the glucose residue discriminating between those deriving from C3 or C4 plants. The analysis of 4, obtained from 3, formed in the beta-glucosidase hydrolysis of salicin 1, reveals in the natural samples from S. purpurea and from Kahlbaum the trend (D/H)4(para) > (DH)3(meta) approximately (D/H)5(meta) > (D/H)6(ortho), the first example of deuterium pattern of an ortho-oxygen-substituted phenylpropanoid. The three samples derived from commercial 1 (Aldrich, Fluka, and Extrasynthese) and the synthetic sample show almost identical deuterium content at positions 4 and 6 (around 153 ppm), whereas for the two meta positions (D/H)3 > (D/H)5 (ca. 162 and 140 ppm, respectively). Product 4, obtained from 3 submitted to acid-catalyzed deuteration, shows different deuterium incorporations in the two meta positions (which are ortho/para to the activating phenolic hydroxyl group), suggesting that possibly the deuterium abundance at the two meta positions may be affected by exchange phenomena with the medium.     

Stable isotope labeling pattern of resveratrol and related natural stilbenes

The stable isotope characterization of resveratrol 1 from Polygonum cuspidatum and of related natural stilbenes 11 and 12 obtained by hydrolysis of the corresponding glucosides 2 and 3 from Rheum is reported. The C(6)-C(2)-C(6) framework of suitably protected derivatives of 1, 2, and 3 has been degraded with ozone to the C(6)-C(1) aldehydes 4, 5, 9, and 10, retaining all hydrogen atoms of the precursors. The natural and synthetic derivatives are characterized and distinguished by natural abundance deuterium nuclear magnetic resonance studies. In the case of anisaldehyde 4 the two series show, as expected, the characteristic difference of the aromatic labeling. The formyl deuterium contents of 4 and 5 from resveratrol are remarkably different, seemingly reflecting the different enrichments existing between positions 3 and 2, respectively, of the phenylpropanoid precursor. The positional delta(18)O values of the extractive materials 1-3 were also determined. In this instance a selective deoxygenation procedure was adopted, leading from 1 to the products 6, 7, and 8. The delta(18)O values of the latter compounds reveal, respectively, those at position 4' and positions 3 and 5 of 1. Similarly, the phenolic products 11 and 12 were converted into 13 and 14. From the delta(18)O values of the single components it is possible to design a detailed map of the oxygen fractionations which characterizes the stilbenes 1-3. In particular, the oxygen present at position 4' of the phenylpropanoid moiety of 1-3 shows delta(18)O values of +11.5, +1.8, and +6.7 per thousand, respectively. Moreover, the phenolic oxygen atom at position 3' of rhapontin 3 shows a value of +11.7 per thousand. The data are compared with those previously obtained on structurally related compounds. These results show the utility of simple chemical degradations in the stable isotope characterization of structurally complex food components.     

1H NMR metabolite fingerprint and pattern recognition of mullet (Mugil cephalus) bottarga

Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate data analysis (MVA) was used to investigate the molecular components of the aqueous extract of samples of bottarga, that is, salted and dried mullet (Mugil cephalus) roe, manufactured in Sardinia (Italy) from mullets of known and unknown geographical provenience. Principal component analysis (PCA) applied to the processed (1)H NMR spectra indicated that samples tend to cluster according to their geographical origin and also on the basis of storage and manufacturing procedures. The most important metabolites that characterized grouping of samples are the free amino acids methionine (Met), glutamate (Glu), histidine (His), phenylalanine (Phe), tyrosine (Tyr), and isoleucine (Ile); trimethylamine (TMA) and dimethylamine (DMA), both biomarkers of degradation; nucleotides and derivatives; choline (Cho) and phosphorylcholine (P-cho); and lactate (Lac).     

High-resolution NMR and diffusion-ordered spectroscopy of port wine

The use of high-resolution NMR and high-resolution diffusion-ordered spectroscopy (DOSY) for the characterization of selected Port wine samples of different ages with the aim of identifying changes in composition is described. Conventional 1D and 2D NMR methods enabled the identification of about 35 compounds, including minor components such as some medium-chain alcohols, amino acids, and organic acids. High-resolution (HR) DOSY extended sample characterization, increasing the number of compounds identified and NMR assignments made, by providing information on the relative molecular sizes of the metabolites present. Port wines of different ages were found to differ mainly in their content of (a) organic acids and some amino acids, (b) an unidentified possible disaccharide, and (c) large aromatic species. The relative amount of these last high Mw aromatics is seen to decrease significantly in the oldest wine, as expected from the known formation and precipitation of anthocyanin-based polymers during red wine aging.     

Authentication of the origin of vanillin using quantitative natural abundance 13C NMR

The use of 13C isotopic distribution as an efficient means to determine the origin of vanillin has been substantiated. Using quantitative 13C NMR, the 13C/12C ratios at all eight carbon positions can be exploited. On a set of 21 samples of vanillin from five different origins, complete discrimination can be achieved. It is shown that, for many purposes, a rapid analysis in which only five sites are used is sufficient. However, improved discrimination using all eight sites is preferable to differentiate between different methods of production from natural ferulic acid or between natural and lignin-derived vanillin on the basis of the 13C/12C ratios characteristic of different origins. The C1 and C8 positions are demonstrated to be the most significant sites for discrimination using principle component analysis. However, aromatic carbon positions make an essential contribution, notably in differentiating between natural and lignin-derived vanillin.     

Consumption of a functional fermented milk containing collagen hydrolysate improves the concentration of collagen-specific amino acids in plasma

Clinical studies have shown that collagen hydrolysate (CH) may be able to protect joints from damage, strengthen joints, and reduce pain from conditions like osteoarthritis. CH is a collection of amino acids and bioactive peptides, which allows for easy absorption into the blood stream and distribution in tissues. However, although various matrices have been studied, the absorption of specific amino acids from CH added to a fresh fermented milk product (FMP) was not studied. The primary objective of the present study was to compare the plasma concentrations of four representative amino acids from the CH (glycine, proline, hydroxyproline, and hydroxylysine) contained in a single administration of a FMP with that of a single administration of an equal amount of neat hydrolyzed collagen. These four amino acids were chosen because they have already been used as markers of CH absorption rate and bioavailability. This was a single-center, randomized open, and crossover study with two periods, which was performed in 15 healthy male subjects. The subjects received randomly and in fasted state a single dose of product 1 (10 g of CH in 100 mL of FMP) and product 2 (10 g of CH dissolved in 100 mL of water) separated by at least 5 days. After administration, the subjects were assessed for plasma concentrations of amino acids and for urine concentrations of hydroxyproline. After FMP administration, mean values of the maximal concentration (Cmax) of the four amino acids were greater than after ingredient administration (p < 0.05). This effect was related to an increased Cmax of proline (p < 0.05). In conclusion, because of their physicochemical characteristics, the fermentation process, and the great homogeneity of the preparation, this milk product improves the plasma concentration of amino acids from CH, that is, proline. The present study suggests an interesting role for FMP containing CH to improve the plasmatic availability of collagen-specific amino acids. Hence, this FMP product could be of potential interest in the management of joint diseases.     

Factors affecting the formation of alkylpyrazines during roasting treatment in natural and alkalinized cocoa powder

The cocoa roasting process at different temperatures (at 125 and 135 degrees C for 3 min, plus 44 and 52 min, respectively, heating-up times) was evaluated by measuring the initial and final free amino acids distribution, flavor index, formol number, browning measurement, and alkylpyrazines content in 15 cocoa bean samples of different origins. These samples were also analyzed in manufactured cocoa powder. The effect of alkalinization of cocoa was studied. Results indicated that the final concentration and ratio of tetramethylpyrazine/trimethylpyrazine (TMP/TrMP) increased rapidly at higher roasting temperatures. The samples roasted with alkalies (pH between 7.20 and 7.92), such as sodium carbonate, or potassium plus air injected in the roaster during thermal treatment, exhibited a greater degree of brown color formation, but the amount of alkylpyrazines generated was adversely affected. The analysis of alpha-free amino acids at the end of the roasting process demonstrated the importance of the thermal treatment conditions and the pH values on nibs (cocoa bean cotyledons), liquor, or cocoa. Higher pH values led to a lower concentration of aroma and a higher presence of brown compounds.     

Identification of amino acids in wines by one- and two-dimensional nuclear magnetic resonance spectroscopy

Amino acids are minor compounds in wines, but they have a profound influence on wine quality, and amino acids composition can be used to differentiate wines according to the vine variety, geographical origin, and year of production. The NMR signals of amino acids in NMR spectra are overlapped by the signals of other compounds present and especially by the signals of dominant compounds such as water, ethanol, and glycerol. In this work we used 1D (1)H and (13)C, 2D homonuclear COSY, TOCSY, and 2D heteronuclear HSQC and HMQC pulse sequences, also with an incorporated WET pulse sequence element that allows the simultaneous suppression of several frequencies. Complete (1)H and (13)C NMR assignments for 17 amino acids commonly present in wine and of gamma-aminobutyric acid at pH 3 have been achieved in wine sample of Sauvignon from the Coastal wine-growing region of Slovenia, vintage 1994.     

Chemical characterization and DNA tracking of Sardinian botargo by Mugil cephalus from different geographical origins

The chemical composition of the Sardinian botargo by Mugil cephalus from different geographical origins was investigated. Fat ( approximately 20%), proteins ( approximately 50%), moisture ( approximately 22%), and salt ( approximately 7%) were measured in ground (G) and whole (W) commercial products. Among the nutritional compounds, omega-3 fatty acids were approximately 8%, squalene was approximately 15 mg/100 g, vitamin E was approximately 8.5 mg/kg, and cholesterol was approximately 300 mg/100 g, on average in both products. Antioxidant properties, assessed by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) test and expressed as Trolox equivalent antioxidant capacity (TEAC), showed quite good activity in extracted oil (0.8-1.1 mmol of Tolox/L). Major constituents in the samples varied noticeably, but only few statistical differences were evidenced between G or W products or between samples from different origins. Principal component analysis (PCA) of random amplified polymorphic DNA (RAPD) and proteins, coupled with both, did not differentiate samples from different origins. On the basis of our results, chemical and molecular data exclude the differentiation of samples from diverse origins.     

Origin of the high-frequency resonances in 1H NMR spectra of muscle tissue: an in vitro slow magic-angle spinning study

High-resolution slow magic-angle spinning (150 Hz) 1H PASS NMR spectroscopy is performed on intact excised rat m. tibialis anterior. Untreated muscles and muscles in vitro incubated in Krebs-Ringers buffer based on deuterium oxide are investigated. In the high-frequency region of the 1H NMR spectra, resonances from H4 (approximately 7.1-7.2 ppm) and H2 (approximately 8.2-8.5 ppm) in histidine are observed. In addition, a resonance appears at 6.7 ppm for the untreated muscles. However, this resonance is absent in muscles following incubation in deuterium oxide. On the basis of its behavior in deuterium oxide combined with supplementary measurements for creatine solutions, the 6.7 ppm resonance is ascribed to the amino protons in creatine. Moreover, the present study demonstrates that the observation of the 6.7 ppm resonance depends on pH, which explains earlier reports stating its occasional appearance. Finally, measurements on solutions of ATP/AMP and histidine indicate that both ATP/AMP and histidine contribute to the resonances at approximately 8.2-8.5 ppm in the 1H NMR spectra of muscle tissue.     

Authenticity assessment of gamma- and delta-decalactone from prunus fruits by gas chromatography combustion/pyrolysis isotope ratio mass spectrometry (GC-C/P-IRMS)

Authenticity assessment of gamma-decalactone (1) and delta-decalactone (2) from peach (Prunus persica var. persica), apricot (Prunus armeniaca), and nectarine (Prunus persica var. nectarina) was performed using gas chromatography-isotope ratio mass spectrometry (GC-IRMS) in the combustion (C) and pyrolysis (P) mode. In addition, commercially available synthetic (nature-identical) 1 and 2 as well as biotechnologically produced samples (declared to be "natural") were characterized by their delta(2)H(V)(-)(SMOW) and delta(13)C(V)(-)(PDB) values. For the Prunus fruits under study, rather narrow ranges of delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data of 1, varying from - 34.6 per thousand to - 38.4 per thousand and -160 per thousand to -206 per thousand, respectively, were obtained. Synthetic references of 1 showed delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data ranging from -27.4 per thousand to -28.3 per thousand and -151 per thousand to -184 per thousand, respectively. Samples of 1 declared to be "natural" exhibited ranges from -28.1 per thousand to -29.2 per thousand and -192 per thousand to -286 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively. For 2 from peach, apricot, and nectarine, delta(13)C(V)(-)(PDB) values ranging from -34.0 per thousand to -37.9 per thousand were determined; the delta(2)H(V)(-)(SMOW) values ranged from -171 per thousand to -228 per thousand. The delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data for synthetic 2 were -28.2 per thousand and -171 per thousand, respectively, that is, similar to those of 2 from "natural" origin, ranging from -27.7 per thousand to -30.1 per thousand and -185 per thousand to -230 per thousand for delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW), respectively. GC-C/P-IRMS allowed clear-cut analytical differentiation of the synthetic and "ex-plant" origin of 1 and 2, whereas narrow ranges of delta(13)C(V)(-)(PDB) and delta(2)H(V)(-)(SMOW) data were found for samples of synthetic and "natural" origin.     

Precolumn phenylisothiocyanate derivatization and liquid chromatography of amino acids in food

A precolumn phenylisothiocyanate derivatization method is described for the determination of amino acids in protein hydrolysates from a wide variety of complex food matrixes, with and without performic acid oxidation pretreatment. Analysis of samples that were not pretreated with performic acid was necessary since this pretreatment destroyed an average of 25% of the histidine and 87% of the tyrosine present in the food samples. This method is rapid and reproducible; coefficients of variation between duplicate analyses of the same food item were less than 5% for a majority of the amino acids. Occasionally, variation between duplicate analyses for histidine and tyrosine was greater than 10%. Recoveries of amino acids added to samples were in the 100% range.     

Thermally generated 3-aminopropionamide as a transient intermediate in the formation of acrylamide

On the basis of the recent findings that "biogenic amines" can also be formed during thermal food processing from their parent amino acids in a Strecker-type reaction, the formation of 3-aminopropionamide, the biogenic amine of asparagine, was investigated in model systems as well as in thermally processed Gouda cheese. The results of model studies revealed that, besides acrylamide, 3-aminopropionamide was also formed in amounts of 0.1-0.4 mol % when asparagine was reacted in the presence of either glucose or 2-oxopropionic acid. Results of a second series of model experiments in which [(13)C(4)(15)N(2)]-asparagine ([(13)C(4)(15)N(2)]-Asn) and unlabeled 3-aminopropionamide were reacted together in the presence of glucose revealed a >12-fold higher efficacy of 3-aminopropionamide in acrylamide generation as compared to asparagine. Both [(13)C(3)(15)N(2)]-3-aminopropionamide and [(13)C(3)(15)N(1)]-acrylamide were formed during [(13)C(4)(15)N(2)]-Asn degradation in a ratio of about 1:4, supporting the idea that 3-aminopropionamide is a transient intermediate in acrylamide formation. In this study, 3-aminopropionamide was identified and quantified for the first time in foods, namely, in Gouda cheese. Although the fresh cheese contained low amounts of 3-aminopropionamide, its concentrations were much increased to approximately 1300 mug/kg after thermal processing. In isotope labeling studies, performed by administering to the cheese [(13)C(4)(15)N(2)]-Asn in a ratio of 1:2 as compared to the "natural" concentrations of asparagine, similar ratios of unlabeled/labeled 3-aminopropionamide and unlabeled/labeled acrylamide were determined. Thus, 3-aminopropionamide could be verified as a transient intermediate of acrylamide formation during food processing.     

Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: insight into factors limiting enzymatic hydrolysis

Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced solid-state NMR spectral-editing techniques as well as 1H-13C two-dimensional heteronuclear correlation NMR (2D HETCOR) were employed. Our results indicated that dilute acid prehydrolysis selectively removed amorphous carbohydrates, increased aromatic CH/other protonated -C═C- and enriched alkyl CH and CH2 components. Cinnamic acids were increased, and proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated in RES. 2D HETCOR experiments indicated a close association between lignin and the residual carbohydrates. Ketones/aldehydes were not detected in the DAP, in contrast to a report in which an appreciable amount of ketones/aldehydes was generated from the acid pretreatment of a purified cellulose in the literature. This suggested that acid pretreatment may modify the structure of purified cellulose more than biomass and that biomass may be a better substrate than model biopolymers and compounds for assessing structural changes that occur with industrial processing. On the basis of NMR and wet chemical analyses, we found the following factors could cause the limitations to the effectiveness of enzymatic hydrolysis: (1) chemical modification of carbohydrates limited the biologically degradable carbohydrates available; (2) cinnamic acids in the residue accumulated; (3) accessibility was potentially limited due to the close association of carbohydrates with lignin; and (4) proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated.     

Compositional relationships between organic matter in a grassland soil and its drainage waters

We present a novel study of the compositional relationships between soil organic components extractable in aqueous extractants and those in sub-soil drainage and surface runoff waters from the soil. The surface soil (0–20 cm) of a stagnogley in long-term grassland was sequentially and exhaustively extracted in aqueous media at pH values of 7, 10.6 and 12.6. Extracts from the soils and their runoff and drainage waters were processed by the procedures of the International Humic Substances Society (IHSS), and fractionated into humic, fulvic, and XAD-4 acids. Elemental, δ¹³C, δ¹⁵N, sugar, amino acids, and solid state CPMAS ¹³C NMR analyses were used to identify similarities and differences between the fractions from the different extracts. There were few differences between the compositions of drainage water samples taken 1 year apart, and these had compositional features similar to those from the more highly oxidized fractions isolated from the soil at pH 7. There were significant differences between the humic components from the drainage waters and isolated from the soil at pH 7 and those of the humic fractions isolated at the higher pH values whose compositions are more clearly related to origins in plants. The compositions of the surface runoff waters indicate origins in transformed plant and animal manures on the soil surface, whereas those of the deep drainage waters originate in more extensively transformed materials, including products of microbial metabolism. The resin technique used in the fractionation allowed the isolation of novel humic acid fractions from the soil extracts, in particular at pH 7 and 12.6. These fractions clearly originated in microbial sources, were rich in saccharides and amino acids (peptides), and low in lignin-derived components.     

Synthesis and fungicidal activity of macrolactams and macrolactones with an oxime ether side chain

Three series of novel macrolactams and macrolactones--12-alkoxyimino-tetradecanlactam, 12-alkoxyiminopentadecanlactam, and 12-alkoxyiminodecanlactone derivatives (7A, 7B, and 7C)--were synthesized from corresponding 12-oxomacrolactams and 12-oxomacrolactone. Their structures were confirmed by 1H NMR and elemental analysis. The Z and E isomers of 7A and 7B were separated, and their configurations were determined by 1H NMR. These compounds showed fair to excellent fungicidal activities against Rhizoctonia solani Kühn. It is interesting that the Z and E isomers of most of the compounds have quite different fungicidal activities. The fact that the compounds have a gradual increase of fungicidal activity in the order of 7A, 7C, and 7B indicated that the macrocyclic derivatives with a hydrogen-bonding acceptor (=N-O-) and a hydrogen-bonding donor (-CONH-) on the ring, and a three methylenes distance (CH2CH2CH2) between these two functional groups, exhibited the best fungicidal activity. The bioassay also showed that 7B not only has good fungicidal activity but also may have a broad spectrum of fungicidal activities.